Currently, international concern is heightened regarding the use of Smallpox virus as a bioterrorism agent. Smallpox is a disease caused by an infection with the variola virus, a member of the genus Orthopoxvirus. The last naturally occurring case of Smallpox was reported in Somalia in 1977. Since recommendations for routine Smallpox vaccination were rescinded in North America and most of Europe in 1971, and the effectiveness of vaccination appears to last only 10 years, much of the world population is currently susceptible to infection. During the Smallpox era, overall mortality rates were approximately 30%. Death usually occurred late in the first week or during the second week of illness and was usually attributed to overwhelming viremia. The virus is highly transmissible from person-to-person and infected individuals may, in turn, infect tens to hundreds of susceptible contacts.
The only acknowledged stockpiles of variola virus, the causative agent of smallpox, are those maintained in the USA and Russia (Henderson, D. A. et al. JAMA 281,2127-2137 (1999)). The recent anthrax attack on the USA, however, has renewed fears that additional stockpiles do exist and could be used as a bioterrorist weapon on a now largely susceptible population. The diagnosis of ordinary-type smallpox was relatively easy when endemic, and was based on the distribution and evolution of the rash. However, in non-endemic regions, smallpox could sometimes be confused with chickenpox which is caused by a herpesvirus (varicella) (Fields' virology, Knipe, D. M., Howley, P. M. (eds)-4th ed., 2001).
Other members of OPV that can cause clinical disease in man and for which discrimination is needed are Monkeypox virus (MPXV), which produces a clinically similar, although usually less severe disease that has, until recently, been restricted to western sub-Saharan Africa (Centers for Disease Control and Prevention. 2003, Morb. Mortal. Wkly. Rep. 52:537-540; Esposito and Fenner, 2002. Poxviruses, p. 2885-2921. In Knipe et al., Fields' virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). Vaccinia virus (VACV) and Cowpox virus (CWPX) can both infect humans, normally resulting in a mild disease (Esposito and Fenner, 2002), although VACV has been known to cause severe, even fatal complications following vaccination (Henderson et al. 1999, JAMA 281:2127-2137). It is unclear if Camelpox virus (CMPX) causes disease in humans but it's genome has recently been recognized as being the most closely related to VARV (Gubser and Smith, 2002, J. Gen. Virol. 83:855-872) and concerns raised over possible genetic manipulation to a human virulent strain.
Ideally, diagnostic tests for OPV must be rapid, sensitive and discriminatory for the OPV that cause significant disease in humans. Very rapid methods have been described such as real-time 5′ nuclease PCR (polymerase chain reaction)(Espy et al., 2002, Mayo Clin. Proc. 77:624-628; Hazelton and Gelderblom, 2003, Emerg. Infect. Dis. 9:294-303; Ibrahim et al., 2003, J. Clin. Microbiol. 41:3835-3839; Ibrahim et al., 1997, Mol. Cell. Probes 11:143-147; Kulesh et al., 2004, J Clin Microbiol. February; 42(2):601-9; Mackay et al., 2002, Nucleic Acids Res. 30:1292-1305; Nitsche et al., 2004, Clin Microbiol. March; 42(3):1207-13) and PCR followed by oligonucleotide microarray hybridization (Ibrahim et al., 1998, Anal. Chem. 70: 2013-2017; Lapa et al., 2002, J. Clin. Microbiol. 40:753-757.) but these techniques require specialized instrumentation that may not always be available. PCR based assays have been described to detect and differentiate OPV infections and when combined with sequencing or restriction fragment length polymorphism (RFLP) analysis of products can offer a high degree of sensitivity and discrimination and can be accomplished in many existing laboratories (Loparev et al., 2001, J. Clin. Microbiol. 39:94-100; Meyer et al., 1997, J. Virol. Methods 64:217-221; Neubauer et al., 1998, J. Virol. Methods 74:201-207; Ropp et al., 1995, J. Clin. Microbiol. 33:2069-2076).
For most laboratories, the lack of positive controls for VARV is a significant concern for both the amplification step and subsequent RFLP analysis. The development of molecular biological tests as diagnostic tools in infectious diseases began, for the most part, after nations had destroyed their VARV stocks or deposited them at the two reference centers. Access to this material is extremely limited and impossible for most organizations making controlled testing procedures difficult or impossible to put in place.
As discussed above, when faced with a potential smallpox or monkeypox outbreak, time is of the essence. Clearly, a quick and easy method of determining if orthopoxvirus is present within a sample and identifying the orthopoxvirus is needed. Diagnostic tests need to be available to rapidly and accurately detect and differentiate these pathogens, especially VARV and MPXV, in clinical material for appropriate public health actions to be initiated: To address this need we have developed rapid and highly sensitive PCR-RFLP assays targeted to the OPV hemagglutinin (HA) and cytokine response modifier B (crmB) genes, complete with synthetic positive controls, suitable for use in most routinely equipped laboratories.